Obesity is frequently associated with insulin resistance and constitutes the major risk factor for non insulin-dependent diabetes mellitus (NIDDM). In recent years, studies have indicated that in obesity, the elevated expression of TNFalpha by adipose cells plays a critical role in the development of insulin resistance. TNFalpha interferes with insulin action by inhibiting the tyrosine kinase activity of the insulin receptor (IR) both in cultured cells and in whole animals. This inhibition of IR signaling appears to be through TNFalpha-induced serine phosphorylation of insulin receptor substrate 1 (IRS-1). However, the molecular components that are involved in this process, especially concerning the proximal signaling events activated by TNF are not known. In this application we propose to address these components by studying the biological functions of TNFalpha mediated through its two identified receptors (TNFR1 and TNFR2) in relation to TNFalpha-induced insulin resistance and other aspects of energy metabolism in whole animals. We will address the role of each receptor in TNFalpha-induced insulin resistance by utilizing mice which carry targeted mutations in TNFR1, TNFR2 and both. We will first characterize the metabolic state of these animals in detail. These will be followed generating obese animals which are deficient for one or both of the TNF receptors. Initially, this will be accomplished by cross-breeding TNFR mutant mice into a genetic model of obesity and insulin resistance (ob/ob mice). The metabolic state of the resulting progeny will be studied with particular focus on glucose homeostasis and insulin action to understand which TNF receptor is primarily involved in this process. In addition, we will investigate the role of each TNF receptor in diet-induced obesity and insulin resistance, during which there is also elevated TNFalpha expression in adipose tissue. These experiments will be followed by reconstituting TNF receptor expression in the TNFR1/TNFR2-deficient mice in a adipose tissue- specific fashion. The aP2 promoter/enhancer region will be used to direct the transgenic expression of TNFR1 or TNFR2 to adipose tissue. The resulting mice will then be compared to both control and TNFR- deficient mice mainly focusing on glucose homeostasis and adipose tissue development in response to dietary or genetic manipulations to induce obesity. These experiments will allow us to study the role of TNF signaling through each TNF receptor in adipose tissue during the development of insulin resistance in vivo.